Life on earth is largely supported by the solar energy captured by the green plastids (chloroplasts) present in land plants and green algae. Chloroplasts are considered to have originated from a photosynthetic prokaryote (a cyanobacterium) that entered into an endosymbiotic relationship with an early eukaryotic progenitor over a billion years ago [Goksoyr J, Nature 214; 1161, 1967; Martin and Müller, Nature 392; 37-41, 1998; Martin and Kowallik, Eur. J. Phycol. 34, 287-295, 1999]. During the course of evolution, majority of the chloroplast genome (plastome) has moved to the nucleus, leaving behind a small but functional genome in the chloroplasts coding key components involved in photosynthesis, transcription and translation machinery. Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes suggested that about 4,500 Arobidopsis nuclear-encoded proteins (˜18% of the total nuclear-coding genes) might have been acquired from the cyanobacterial ancestor of the plastids [Martin W, Proc. Natl. Acad. Sci. USA 99, 12246-12251, 2002].
Such a large scale migration of organellar genome to the nucleus has necessitated the development of an elaborate protein import mechanism(s) from the cytosol into chloroplasts for their ultimate function by crossing the double membrane envelope that surrounds each plastid. The nuclear-encoded proteins destined to plastids are made as preprotiens having usually a cleavable N-terminal amino acid extension (transit peptide) that target the protein into plastids via the general protein import pathway. The general protein import machinery of the plastids consists of protein complexes present in the outer (Toc) and inner (Tic) membranes of chloroplasts [Robson and Collinson, EMBO Reports 7, 1099-1103, 2006].
A comparative molecular structure and functional analysis indicated a high degree of similarity between the subunits present in the Toc and Tic complexes and with the prokaryotic protein secretion pathway SecYEG complexes. Indeed the complete genome sequence of Arabidopsis and rice revealed the presence of all the genes coding for the key components of SecYEG pathway involved in protein translocation in bacteria: SecA, SecY and SecE. Also genes coding for SecA and SecB known to be involved in the secretion of proteins through SecYEG pathway in bacteria are present in the nuclear genomes of land plants. Based on the genetic evidences and the similarity of the proteins at the molecular level, it has been postulated that the same protein export mechanism that functioned originally to secrete the proteins in bacteria might have now been operating in the opposite direction to import proteins from the host cell into the chloroplasts to cross the double membrane chloroplast envelop after suitable modification(s) during the course of evolution [McFadden, Curr. Opinion in Plant Biol. 2, 513-519, 1999].
In the present day, land plant plastids, it is not known whether the protein export mechanisms that were originally operating before the endosybiosis are still functional and a foreign protein expressed in chloroplasts can be secreted into the cytoplasm of plant. Therefore, one of our main goals is to understand the protein trafficking from chloroplasts into the cytoplasm and find out the transport mechanisms present in the chloroplasts that can export foreign proteins synthesized in the plastids into cytoplasm of the cell. Such an understanding not only throw light on the evolutionary changes that took place in the basic biological process in the eukaryotic cell over millions of years, but also provide opportunities to engineer plastid genome to express foreign proteins with functions outside the chloroplasts that include creation of post translational modifications such as glycosilation, a major limiting step in chloroplast genetic engineering but very important in biotechnological application. In addition, the implications of these findings in relation to the biosafety of genetically modified organisms (GMOs) in the context of environment and biodiversity are discussed.
Chloroplasts are generally believed to have originated from a photosynthetic cyanobacterium-like prokaryote. During the course of evolution spanning over a billion years the prokaryotic organism has entered into an endosymbiontic relationship with the early eukaryotic cell. As a consequence majority of the chloroplast genome has moved to the nuclear genome and the corresponding proteins synthesized in the cytoplasm are imported back into plastids crossing the double membrane envelop through protein import mechanisms. It is not known whether the original protein export mechanisms that were operating before the endosymbiosis are still functional in the present day chloroplasts or not. The present invention, for the first time, the presence of evolutionarily conserved and fully functional prokaryotic-type protein secretary pathway in chloroplasts of higher plants, operating just in opposite direction to the well established general protein import pathway. The implications of the newly discovered functionally usable prokaryotic-type secretary pathway in chloroplasts in the evolution of eukaryotic cell, for biotechnological applications and in the biosafety of transgenics in the context of environment and biodiversity are discussed.
Biosafety is a serious public concern today due to large scale deployment of GMOs into the environment in several countries across the globe and this is another important area where the present invention is expected to have a major impact. As plastids are inherited maternally in most crops, chloroplast genetic engineering is considered as a better strategy to contain foreign gene flow to untransformed plants and wild relatives [Daniell H, Nature Biotechnology 20, 581-586, 2002; Ruf et. al., Proc. Natl. Acad. Sci. USA 104, 6998-7002, 2007; Savb and Maliga, Proc. Natl. Acad. Sci. USA 104, 7003-7008, 2007], a perceived threat to ecological imbalance and a major challenge for the conservation of biodiversity. The application of the chloroplast genetic engineering in plant biotechnology is limited so far to only those few genes that have or can function from within the chloroplasts [Maliga P, TRENDS in Biotechnology 21, 20-28, 2003; Daniell et. al., TRENDS in Plant Sci. 6, 219-226, 2001].
The present invention discloses that any foreign protein having function(s) out side the chloroplast can now be expressed in chloroplasts and target the protein to its site of function. With the use of appropriate targeting peptides it is possible in future to target the chloroplast expressed recombinant proteins in to any sub-cellular compartment in the cell. This would essentially allow one to contain the transgene flow by integrating the foreign gene into plastid genome that would improve the biosafety of the GMO's to the environment to a very high level and at the same time use the gene function(s) in any compartment of the cell.